The present invention relates generally to methods and reagents used for the determination of ligands in biological fluids. In particular, the present invention relates to a novel class of 4'-methyl substituted fluorescein conjugates useful as tracer reagents in fluorescence polarization immunoassays.
Drug levels in serum samples can be determined through competitive binding immunoassays. Competitive binding immunoassays for measuring the concentration of an analyte (also referred to as a ligand), such as a drug in a test sample are based on the competition between a ligand in a test sample and a labeled reagent, referred to as a tracer, for a limited number of receptor binding sites on antibodies specific to the ligand and tracer. The concentration of ligand in the sample determines the amount of the tracer that will specifically bind to an antibody. The amount of tracer-antibody conjugate produced can be quantitatively measured and is inversely proportional to the quantity of ligand in the test sample.
Examples of ligands or drugs measurable by the methods of the present invention include steroids such as estriol, adrenocorticotropic hormone (ACTH), estrone, cholesterol, estradiol, cortisol, testosterone, progesterone, chenodeoxycholic acid, digoxin, cholic acid, digitoxin, deoxycholic acid, lithocholic acids and the ester and amide derivatives thereof; vitamins such as B-12, folic acid, thyroxine, triodothyroxine, histamine, serotonin, prostaglandins such as prostaglandin E(PGE), prostaglandin F(PGF), prostaglandin A(PGA); anti-asthamatic drugs such as theophylline; antineoplastic drugs such as doxorubicin and methotrexate; antiarrhythymic drugs such as disopyramide, lidocaine, procainamide, propranolol, quinidine, N-acetyl procainamide; anticonvulsant drugs such as phenobarbital, phenytoin, primidone, valporoic acid, carbamazepine, flecainide and ethosuximide; antibiotics such as penicillins, cephalosporins, erythromycin, vancomycin, gentamicin, amikacin, chloramphenicol, streptomycin and tobramycin; antiarthritic drugs such as salicylates; antidepressant drugs including tricyclics such as nortriptyline, amitriptyline, imipramine and desipramine; and the like as well as the metabolites thereof. In addition, drugs may include drugs of abuse such as morphine, heroin, hydromophone, oxymorphone, methadone, codeine, hydrocodone, dihydrocodeine, dihydrohydroxy codeinone, dextromethorphan, phenazocine, benzoyl ecgonine, tetrahydro-cannabinoids (THC), barbiturates, benzodiazepine, lysergic acid diethylamide (LSD), propoxyphene, phencyclidine, amphetamines, methaqualone, and their metabolites may be measured in accordance with the methods of the present invention. In addition, environmental pollutants may be measured in accordance with the methods of the present invention. Examples of environmental pollutants include pesticides, herbicides, insecticides, fungicides such as polychlorinated biphenyls (PCBs), atrazine, simazine, terbutryn, s-triazines, amitrole, trifluralin, nortflurazon, permethrin, cypermethrin, paraquat, alachlor, metolachlor, chlorsulfuron, phenylurea herbicides, aldrin, chlordane, endosulan, parathion, dioxins, 2-aminobenzimidazole, pentachlorophenol, benzo-.alpha.-pyrene, polyaromatic hydrocarbons (PAHs) and their metabolites.
Fluorescence polarization (FP) is well known in the immunoassay field as providing a quantitative means for measuring the amount of tracer-antibody conjugate produced in a competitive binding immunoassay (see Biochem.Biophys.Res.Comm. 5:299, 1961). In general, fluorescent polarization techniques are based on the principle that a fluorescein labeled compound when excited by linearly polarized light will emit fluorescence having a degree of polarization inversely related to its rate of rotation.
In fluorescence polarization immunoassays (FPIA), fluorescence polarization is a reproducible function of the ligand or drug concentration, and thus is suitable for the quantitative determination of ligand or drug concentrations in serum for the purpose of therapeutic drug monitoring. When tracer, serum containing antibodies specific for the drug to be measured, and drug-free patient serum are mixed together, most of the tracer binds to the antibodies. As a result, when the bound tracer is excited with polarized light at 489 nm, the light emitted at 520 nm remains highly polarized. However, if drug is present in the patient sample, the drug will compete with the tracer for binding to the antibodies. Thus, more of the tracer will remain unbound and the emitted light is depolarized.
An FPIA according to the present invention can be any type of automated or manual FPIA. Preferably the FPIA is carried out on the automated COBAS FARA II.RTM. chemistry system (Roche Diagnostic Systems, Inc., Branchburg, N.J.) which can measure the binding of fluorescein labeled drug (the tracer) to specific antibodies (see Dandliker and Feigen, Biochem. Biophys. Res. Comm. 5:299, 1961).
In FPIA, the results can be quantified in terms of millipolarization units (mP) from which a calibration curve can be determined and the span can be calculated. The span is the difference (or delta) between the maximum and minimum binding as measured in mP units (delta mP) of the tracer to the antibody as the free drug competes with the bound tracer for antibody binding sites. A larger span provides for better precision in FPIA. The polarization of fluorescence decreases in a regular manner as the concentration of the analyte increases. The higher the delta value, or span, the better the precision and sensitivity of the assay. The concentration of drug in the sample can be determined by comparison to a standard calibration curve.
Several fluorescein derivatives from which fluorescein labelled compounds, or tracers, can be prepared are known and are commercially available. The majority of fluorescein derivatives are derived from the 5 or 6 position of fluorescein (also referred to as isomer I for the 5 position and isomer II for the 6 position) and include 5 or 6-N-hydroxysuccinimidylcarboxyfluorescein, 5-aminomethylfluorescein and 5-or 6-dichloro-1,3,5-triazin-2-ylaminofluorescein (DTAF).
Fluorescein derivatives synthesized out of the 4' position of fluorescein are also known. For example, 4'-aminomethylfluorescein is useful as a nucleophile for coupling to drug derivatives or ligands bearing a carboxylic group (see U.S. Pat. No. 4,614,823 and U.S. Pat. No. 4,510,251). The amine group of the fluorescein reacts with a carboxy group of an analyte to form a peptide bond. However, this method does not allow an analyte containing a cyclic ring to be attached directly at the 4'-methyl carbon atom.
Therefore, it is an object of the present invention to provide a fluorescein derivative which can react directly to cyclic amines to provide a cyclic linker between the fluorescein molecule and the analyte.
More particularly, it is an object of the present invention to prepare an improved 4'-methyl substituted fluorescein derivative modified with a linking group which can be readily conjugated to a ligand having a nucleophilic center. It is also an object of the present invention to prepare a 4'-methyl substituted fluorescein derivative which can be readily conjugated to a ligand modified with a linking group having a nucleophilic center.
Further, it is an object of the present invention to prepare a 4'-methyl substituted fluorescein derivative containing a leaving group which can be readily displaced with an amino group, thereby facilitating the linking of the fluorescein to a drug derivative. The 4'-methyl substituted fluorescein itself can be modified with the amino group, preferably by reaction with a cyclic amine. Alternatively, the amino group can be part of a drug derivative to which the 4'-methyl substituted fluorescein can be conjugated, most preferably a drug derivative having a cyclic amine-containing linker.
A further object of the present invention is to provide a 4'-methyl substituted fluorescein derivative which can be used to prepare a fluorescein tracer having a larger dynamic span which leads to a more precise and sensitive performance in the FPIA.